Combinatorial Engineering Of Transcriptional Regulatory Elements To Construct Efficient Promoters In The Methylotrophic Yeast Pichia Pastoris

With the development of genetic engineering technology,protein expression has become the main way of industrial production.The methylotrophic yeast Pichia pastoris is widely used in the scientific research and industrial production due to the advantages of its capabilities in rapid growth,post translational modification,and high protein expression.The most commonly used promoter of Pichia pastoris to express heterologous proteins is the promoter of the alcohol oxidase 1 gene(hereafter named Paox1),which can rapidly activate gene transcription when methanol is used as the sole carbon source.However,there are still some problems in the Pichia pastoris protein expression system:On one hand,the activity of the aoxl promoter remains to be further strengthened to achieve a higher level of protein expression;on the other hand,methanol is toxic,flammable and explosive,and is not suitable in the production of protein in food and medicine fields,wherein nontoxic carbon sources such as glucose and glycerol need to be applied.Therefore,the engineering of aoxl promoter activity is very important.In addition,with the rapid development of synthetic biology in recent years,the construction of an expression system invovling artificial transcription factors and synthetic promoters in Pichia pastoris has also become one of the research hotspots.It will play an important role in reprogramming cellular mechanisms,and further to benefit human health,food,medicine and energy issues.The main work can be summarized in the following three parts:firstly,we constructed the strains overexpressing the positive transcriptional regulators Mxrl and Mitl of the aoxl promoter,respectively,and analyzed the activities of aoxl promoter in thesestrain under the non-inducing carbon sources,inducing carbon source and the mixed carbon source;secondly,we performed engineering towards the cis-elements present in the aoxl promoter and analyzed the activities of the resulting mutant aoxl promoters under the inducing and non-inducing carbon sources with egfp as a reporter gene.finally,an expression system based on artificial transcription factors and synthetic promoters was constructed in Pichia pastoris and was further optimized in Saccharomyces cerevisiae.The main results are presented as follows:1.The recombinant strains OEMxr1 and OEMitl were constructed to overexpress the transcriptional factors Mxrl and Mitl respectively.Overexpression of the two factors significantly increased the activity of the aoxl promoter under the inducing carbon source,and partially relieved the carbon catabolite repression of the aoxl promoter under the non-inducing carbon source.The overexpression of Mxrl and Mitl were driven by the strong GAP promoter in Pichia pastoris GS115,and the activity of aoxl promoter in the overexpressed strain was detected by the analyses of cell sensitivity towards 2-DG,oxidase activity,and transcriptional expression of aoxl.The results show that the overexpression of Mxrl and Mitl can significantly enhance the aoxl promoter activity under the inducingcarbon source,and can partially relieve the carbon catabolite repression of the aoxl promoter under the non-inducing carbon sources including glucose and glycerol as well as the mixed carbon source of methanol and glycerol.2.The aoxl promoter sequence was modified by eliminating the putative binding sites ofthe mutant repressor Nrgl or icreasing the nucleotide motifs bound by the positive transcriptional regulator Mxrl and Mitl.The activity of the modified aoxl promoter on methanol was significantly enhanced,and the effect was reinforced in combination with overexpression of Mxrl or Mitl.Based upon the systematic analyses of the cis-acting elements on the aoxl promoter,we eliminated the putative binding sites of the negative regulator Nrgl on aoxl promoter(resulting the mutant promoter mutPaox1),and increased the Mxrl binding motifs and the putative Mitl binding motifs(resulting the mutant promoter MBS-Paox1),and further analyzed the activities of the mutant promoters using egfp as a reporter gene.The results showed that the activity MBS-Paox1 in Pichia pastoris GS115 was significantly higher than that in wild type strain under methanol inducing condition.When MBS-Paox1 was introduced into the Mxrl-overexpressing strain,the expression level of the reporter gene was 3 times as high as that of the wild type promoter,which showed that its activity was further enhanced under the methanol induction condition.When MBS-Paox1 was introduced into the Mitl-overexpressing strain,it activity was further improved under glycerol or glycerol-methanol mixed carbon sources.In order to detect the practicability and effectiveness of the expression system,we used MBS-Paox1 to express an alpha-galactosidase from the fungus Talaromyces emersonii in the Mxr1-or Mit1-overexpressing strains.The results show that the expression level of the alpha-galactosidase is significantly improved than that observed in the wild type.3.An expression system involving artificial transcription factors and synthetic promoters was successfully constructed in P.pastoris,and was further optimized in Saccharomyces cerevisiae.Based on the pPICZaA,both of artificial transcription factor ZF expression cassette PGAP-ZF-TT and synthetic promoter expression cassette ZF BD-miniPaox1-yegfp-TT,were transformed into Pichia pastoris GS115.The results of fluorescence analysis showed that the transcriptional ability of the synthetic promoter was 3 to 4 times higher than the basic transcriptional level under the activation of artificial transcription factors.We put the artificial zinc finger transcription factor ZF and synthetic promoter reporter gene expression cassette into Saccharomyces cerevisiae to further optimize the design components of the transcriptional expression system,and preliminarily constructed a dual transcriptional regulatory system involving the repressor protein Lacl.